Figure 1.
Consistent generation of sensory neurons from control and SMA iPSCs.
(A) Control and SMA iPSCs acquire a Tuj1+ (green)/peripherin+ (red) sensory neuron phenotype by 2 weeks of differentiation that remained consistent through 6 weeks of differentiation. Nuclei are labeled with Hoechst nuclear dye (blue). Quantification of the Tuj1+ population (B) and peripherin+ population (C) show no significant difference in neuronal differentiation efficiency between control and SMA iPSCs at any time point. (D) At 6 weeks of differentiation there was no difference in neurite length between control and SMA iPSC-derived sensory neurons. (E) Ratiometric live cell calcium imaging showed a significant reduction in calcium response to KCl depolarization in the SMA iPSC-derived sensory neurons at 2 weeks of differentiation compared to controls. Representative imaging traces of at least 30 individual cells are shown for (F) one control iPSC line and (G) one SMA iPSC line. KCl indicates the time at which the depolarizing stimulus was added to the cultures. n.s. = not significant by ANOVA (B and C) or by Student’s t-test (D). ***p = 0.0008 by Student’s t-test (E). Scale bar = 50 µm.
Figure 2.
Generation of nociceptive neurons from control and SMA iPSCs.
(A) Control and SMA iPSCs generate nociceptive neurons as indicated by NTRK1 (red) and Tuj1 (green). Nuclei are labeled with Hoechst nuclear dye (blue). (B) There was no difference in neuron differentiation between control and SMA iPSC cultures at either 4 or 6 weeks of differentiation. n.s. = not significant by ANOVA. Scale bar = 50 µm.
Figure 3.
Generation of proprioceptive neurons from control and SMA iPSCs at 6 weeks of differentiation.
(A) Both control and SMA iPSCs generate proprioceptive neurons as indicated by parvalbumin (red) and Tuj1 (green). Nuclei are labeled with Hoechst nuclear dye (blue). (B) There was no difference in parvalbumin+neuron number between control and SMA iPSC cultures. n.s. = not significant by t-test. Scale bar = 20 µm.
Figure 4.
Peripheral glial cells are present in the sensory neuron cultures.
GFAP+ glial cells (green) are simultaneously generated during the sensory neuron differentiation in both control and SMA iPSC cultures. Nuclei are labeled with Hoechst nuclear dye (blue). Scale bar = 50 µm.
Figure 5.
The presence of SMA iPSC-derived sensory neurons does not induce motor neuron loss at 4, 6, and 8 weeks of differentiation.
(A) Co-culture of control iPSC-derived SMI-32+ (green) motor neurons (MN) with either SMA iPSC-derived peripherin+ (red) sensory neurons (SN) or control iPSC-derived peripherin+ (red) SNs does not induce motor neuron loss. Nuclei are labeled with Hoechst nuclear dye (blue). Quantification is shown in B and C. n.s. = not significant by ANOVA. Scale bar = 50 µm.
Figure 6.
The presence of SMA iPSC-derived sensory neurons (SN) does not induce loss of afferent innervation on control iPSC-derived motor neurons (MN) over 4–8 weeks in culture.
(A) VGlut1+ puncta (green) can be identified on both the cell soma and neurites of SMI-32+ control iPSC-derived MNs (red), as indicated by white arrows regardless of being co-cultured with control or SMA iPSC-derived SNs. Nuclei are labeled with Hoechst nuclear dye (blue). There is a trend toward reduced VGlut1+ puncta on both the cell soma (B) and neurites (C) of control iPSC-derived MNs in the presence of SMA iPSC-derived SNs at 4, 6, and 8 weeks in co-culture, but this trend did not reach significance. n.s. = not significant by ANOVA or by Student’s t-test for each time point. Scale bar = 20µm.